E2 sandwich method rabbit monoclonal antibody mAb9 or antigen binding fragment thereof, and preparation method and application thereof

Abstract

The invention belongs to the technical field of immunodetection, and discloses an E2 sandwich-method rabbit monoclonal antibody mAb9 or an antigen binding fragment thereof as well as a preparation method and application of the E2 sandwich-method rabbit monoclonal antibody mAb9 or the antigen binding fragment thereof. The E2 sandwich method rabbit monoclonal antibody mAb9 or an antigen binding fragment thereof comprises a light chain variable region VL and a heavy chain variable region VH, the light chain variable region VL comprises complementary determining regions LCDR1, LCDR2 and LCDR3, and the heavy chain variable region VH comprises complementary determining regions HCDR1, HCDR2 and HCDR3. The monoclonal antibody can be combined with an E2 compound in a high-specificity mode, is suitable for double-antibody sandwich immunodetection and is applied to an immunodetection kit prepared through double-antibody sandwich ELISA or a chemiluminescence method, a double-antibody sandwich chemiluminescence platform is used for detecting an E2 standard antigen, the detection sensitivity is lower than 10 pg / mL, a clinical sample is detected through a magnetochemiluminescence method, and the detection sensitivity is lower than 10 pg / mL. Within the sample range of 0-15000 pmol / L, the correlation with clinical comparison is good, and clinical management is provided for female health and male health.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, specifically to an E2 sandwich method rabbit monoclonal antibody mAb9 or its antigen-binding fragment, its preparation method and application. Background Technology

[0002] E2, short for estradiol, is a type of estrogen derived from cholesterol. Primarily secreted by the ovaries in women (or the testes and adrenal glands in men), it is the most potent and important estrogen in the human body, exerting a wide-ranging influence on the reproductive system, bone metabolism, and cardiovascular health. Its core physiological functions include its effects on the reproductive system and secondary sexual characteristics, such as promoting endometrial thickening, follicle development and ovulation, and maintaining the menstrual cycle; promoting breast development, pelvic widening, and subcutaneous fat accumulation, and maintaining female secondary sexual characteristics; and its effects on the skeletal and cardiovascular systems, such as promoting calcium deposition, inhibiting bone resorption, and maintaining bone density. The decline in E2 levels after menopause is a key cause of osteoporosis; it also improves vascular endothelial function, promotes vasodilation, has antioxidant and anti-inflammatory effects, and provides cardiovascular protection; and it affects skin, mood, and cognitive function.

[0003] E2 is an important clinical indicator for assessing women's and men's health. It can also aid in the diagnosis of estrogen-secreting tumors and in tumor diagnosis and detection. This includes assessing ovarian function and menstrual cycles, assisted reproduction and infertility treatment, diagnosing precocious puberty or developmental abnormalities, and assessing menopausal status.

[0004] Currently, mass spectrometry is considered the "gold standard" for E2 detection. However, mass spectrometry requires complex and time-consuming sample pretreatment procedures, and clinical applications demand high levels of automation, making large-scale application difficult. Therefore, immunoassay is commonly used clinically. Immunoassay is simple to operate and rapid, making it a feasible alternative to mass spectrometry for small molecules. Since small molecules lack antigenic epitopes, competitive immunoassays are commonly used in clinical practice. Competitive immunoassays suffer from low sensitivity, poor specificity, a high risk of false positives, and complex processes.

[0005] To address the technical challenges of competitive methods in small molecule clinical applications, there is an urgent need to develop highly specific and sensitive monoclonal antibodies for the E2 sandwich method. This would overcome the limitations of traditional competitive methods and make it crucial to achieve E2 detection using a double-antibody sandwich method. Summary of the Invention

[0006] To overcome the aforementioned technical problems, this application provides a rabbit monoclonal antibody mAb9 or its antigen-binding fragment prepared by the E2 sandwich method with high specificity and affinity, and its application in immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence method, providing clinical management for women's and men's health.

[0007] In one aspect, this application provides an E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment, comprising a light chain variable region VL and a heavy chain variable region VH. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.1 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.1. The amino acid sequence of LCDR2 is DAS (SEQ ID NO.2) or a variant having an amino acid substitution, deletion, or addition compared to DAS. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.3; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is the sequence shown in SEQ ID NO.4 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.4. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.5 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6 or a variant with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO.6; Furthermore, the E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment can specifically bind to E2 hormone.

[0008] Further, the light chain variable region VL comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.7, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified; the heavy chain variable region VH comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.8, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified.

[0009] Furthermore, amino acid sequences with 95%, 96%, 97%, 98%, or 99% identity.

[0010] Furthermore, the light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.8.

[0011] Furthermore, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

[0012] Secondly, this application also provides a biomaterial selected from polynucleotides, carriers, or cells. The polynucleotide encodes the E2 sandwich antibody mAb9 or its antigen-binding fragment as described in the first aspect; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the vector, or is capable of expressing the E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment as described in the first aspect.

[0013] Furthermore, the cells are eukaryotic cells, preferably mammalian cells, more preferably 293 cells or CHO cells.

[0014] Thirdly, this application also provides a method for preparing the E2 sandwich method rabbit monoclonal antibody mAb9 or its antigen-binding fragment as described in the first aspect, comprising culturing cells as described in the second aspect, wherein the cells are obtained by transforming the cells with a polynucleotide encoding a polynucleotide comprising the E2 sandwich method rabbit monoclonal antibody mAb9 or its antigen-binding fragment, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprising co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0015] Fourthly, this application also provides the application of the E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment described in the first aspect, or the biological material described in the second aspect, wherein the application is selected from one or more of the following: 1) Detection of E2 hormone for purposes other than disease diagnosis and treatment; 2) Prepare immunoassay products for detecting E2 hormone; 3) Used for purifying E2 hormone; 4) Prepare products for purifying E2 hormone.

[0016] In this application, the application is not for diagnostic or therapeutic purposes. Specifically, it can help women understand the natural trends in their estrogen levels, providing an objective reference for whether hormone replacement therapy (HRT) or other lifestyle interventions are needed. In processes such as in vitro fertilization (IVF), where frequent monitoring of serum E2 levels is required to assess follicle development and endometrial receptivity, rapid and sensitive antibody detection technology can be used for immediate testing in laboratories or clinics to help reproductive specialists adjust medication regimens.

[0017] Fifthly, this application also provides an E2 hormone immunoassay product, wherein the immunoassay product comprises the E2 sandwich antibody mAb9 or its antigen-binding fragment as described in the first aspect or the biological material as described in the second aspect.

[0018] Furthermore, the E2 hormone immunoassay product is a reagent or kit for detection using enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), immunoprecipitation, and magnetic microparticle chemiluminescence immunoassay.

[0019] Compared to existing technologies, the E2 sandwich rabbit monoclonal antibody mAb9 of this application can bind to E2 with high specificity and has high affinity, with an affinity constant Ka reaching 5 × 10⁻⁶. 9 L / mol. The E2 sandwich rabbit monoclonal antibody of this application can also be used to prepare various immunoassay kits for detecting E2, especially for use in immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence methods. The double-antibody sandwich chemiluminescence platform for detecting E2 standard antigen has a detection sensitivity of less than 10 pg / ml. Magnetochemiluminescence detection of clinical samples shows good correlation with clinical R values ​​within the sample range of 0-15000 pmol / L. Attached Figure Description

[0020] Figure 1 This is an electrophoresis diagram of the full-length amplification products of the mAb9 heavy and light chains, where M is the DNA molecular weight marker.

[0021] Figure 2 The mAb9 sandwich ELISA method was used to detect cross-linked proteins and target antigen samples. The vertical axis represents the detected OD value.

[0022] Figure 3 The standard curve was used to detect E2 standard antigen using mAb9 magnetic microparticle chemiluminescence immunoassay. The x-axis represents the concentration of E2 standard antigen (pg / mL), and the y-axis represents the detected luminescence value. The R-squared value of the standard curve is shown. 2 =0.9938, linear detection range 0-5 ng / mL, derive the sample concentration calculation formula: y = -0.0252x 2+253.18x-12198.

[0023] Figure 4 The standard curve was used to detect clinical samples using mAb9 magnetic microparticle chemiluminescence immunoassay. The x-axis represents the free E2 concentration (pmol / L) of the clinical samples, and the y-axis represents the detected luminescence value. The R-value of the standard curve is... 2 =0.9601, linear detection range 0-15000pmol / L, derived sample concentration calculation formula: y=25.68x-11463. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.

[0025] 1) Preparation of immunogens

[0026] The E2 complex was prepared with an immunogen purity of over 90%, meeting the purity requirements for preparing monoclonal antibodies.

[0027] 2) Animal immunization The E2 complex prepared above was emulsified with complete Freund's adjuvant at a 1:1 volume ratio and administered subcutaneously to approximately 2 kg New Zealand white rabbits at a dose of 800 μg / rabbit. A second immunization was performed two weeks later, emulsified with incomplete Freund's adjuvant at a 1:1 volume ratio, at a dose of 400 μg / rabbit. Tail blood was collected after both immunizations and serum titers were determined using a serially diluted ELISA method. The OD450 at an ELISA titer of 128,000 was considered to be greater than 1.0. Based on the results, it was determined whether to collect PBMCs or continue immunization. Rabbits with the highest antibody titers were selected for PBMC collection.

[0028] 3) PBMC isolation, specific B cell sorting, and clonal recombination The rabbit was placed supine on the operating table. The fur around the heart was trimmed, and the skin was disinfected with alcohol. The area with the most prominent heartbeat was selected and punctured with a 50mL syringe. Blood flowed into the syringe immediately after the needle entered the heart. The needle was quickly withdrawn after obtaining the required amount of blood. The whole blood in the syringe was transferred into a sterile 50mL tube and mixed with an equal amount of PBS. The mixture was then slowly added dropwise to the lymphocyte separation medium. The mixture was centrifuged at 400×g for 30 minutes at room temperature. After centrifugation, the liquid surface separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (i.e., a mononuclear cell layer), a separation medium layer, and a red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium, thus obtaining rabbit PBMCs.

[0029] Antigen-specific B cells were further sorted from rabbit PBMCs and cultured. Positive clones were selected from the B cell supernatant using antigen-coated ELISA plates. Cells from positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. The full-length light and heavy chain sequences of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones. Rabbit monoclonal antibody expression vectors were constructed using clonal recombination methods, and the sequences were confirmed by sequencing. The results of the amplified full-length PCR products are shown below. Figure 1 .

[0030] 4) Preparation and purification of monoclonal antibodies To obtain multiple rabbit monoclonal antibodies recognizing the E2 small molecule, the heavy and light chain genes of rabbit monoclonal antibodies were loaded into an expression vector. The plasmid was transfected into KEK293 cells, and after 120-144 hours, the culture supernatant contained recombinant rabbit monoclonal antibodies recognizing the E2 small molecule. The cell suspension was collected, the supernatant was obtained by centrifugation, and the antibody was purified by affinity chromatography. The concentration of the purified monoclonal antibody was determined by the BCA method, and then aliquoted, lyophilized, and named rabbit monoclonal antibody mAb9.

[0031] 1) Specificity identification of rabbit monoclonal antibodies.

[0032] Indirect ELSA was used for detection. The ELISA plate was coated with cross-linked protein and E2 complex antigen at a concentration of 1 μg / mL and incubated overnight at 4°C. The plate was then blocked with PBST containing 1% BSA. 10 μg / mL of PBST was added. 4 The purified rabbit monoclonal antibody was diluted 10 times and reacted at 37°C for 50 min. The plate was washed 3 times with PBST, HRP-goat anti-rabbit IgG secondary antibody was added, and the plate was reacted at 37°C for 50 min. The plate was washed 5 times with PBST, TMB was added for color development for 10 min, stop solution was added, and the A450 was measured by microplate reader.

[0033] Figure 2 The results showed that the cross protein reacted negatively with the rabbit monoclonal antibody mAb9, with OD450 values ​​less than 0.1; the E2 complex reacted positively with the mAb9 antibody, with OD values ​​much higher than those of the cross protein, indicating that the E2 sandwich method rabbit monoclonal antibody of this application specifically recognizes the E2 complex.

[0034] 2) Determination of affinity constant of rabbit monoclonal antibody Affinity constant (Ka) was determined using a non-competitive ELISA method.

[0035] Coating: Dilute the antigen with carbonate buffer to concentrations of 1, 0.5, 0.1, and 0.05 μg / mL, add 100 μL / well to a 96-well microplate for coating, and incubate at 4°C for 24 h.

[0036] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h.

[0037] Add monoclonal antibody: Wash the plate 4 times with PBST, serially dilute the rabbit monoclonal antibody with carbonate buffer starting at 100 μg / mL, add 100 μL to each well, and incubate at 37°C for 2 h.

[0038] Add enzyme-labeled secondary antibody: Wash the plate 4 times with PBST, add 100 μL of HRP enzyme-labeled goat anti-rabbit Ig secondary antibody diluted 1:10000 to each well, and incubate at 37℃ for 30 min.

[0039] Color development and termination: Wash the plate 4 times with PBST, add 100 μL of substrate color development solution to each well, and react at 37℃ in the dark for 15 min; add 50 μL of 1.0 mol / L H2SO4 stop solution to each well to terminate the reaction.

[0040] Detection: The absorbance value at a wavelength of 450 nm (A450nm) was measured.

[0041] Plotting the logarithm of antibody concentration on the x-axis and OD value on the y-axis, an S-shaped curve was constructed. The calculated affinity constant Ka for the E2 sandwich monoclonal antibody mAb9 was 5 × 10⁻⁶. 9 L / mol.

[0042] 3) Sandwich antibody pairing

[0043] To select the optimal combination of coating and detection antibodies, E2-binding protein was coated onto an ELISA plate and incubated overnight at 4°C. The next day, the plate was removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed three times with PBST. 100 μL of the E2 complex (20 ng / mL) was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, the plate was removed, washed three times with PBST, and HRP-labeled rabbit monoclonal antibody mAb9 was added as the detection antibody. The plate was incubated at 37°C for 1 hour. After washing five times with PBST, TMB substrate was added, and the plate was incubated at 37°C for 10 minutes. Stop solution was added, and the OD450 reading was measured using a microplate reader. Based on the OD values ​​of the samples and the background value of the negative control, the most ideal antibody pair was selected. The pairing screening results are shown in Table 1.

[0044] Table 1. Results of antibody pairing assay

[0045] Therefore, the antibody mAb9 involved in this application is optimal for sandwich assay.

[0046] Using the recombinant plasmid of the antibody as a DNA template, sequencing primers for the light chain variable region and heavy chain variable region were designed based on the vector sequences at the 5' ends of the light and heavy chains on the template. Sequencing was performed using an ABI 3730 sequencer. The nucleotide sequences of the light and heavy chain variable regions of the rabbit monoclonal antibody were obtained by sequencing.

[0047] Using the internet and the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the nucleotide sequences of the light chain variable region and the heavy chain variable region were analyzed. The amino acid sequence of the light chain variable region of rabbit monoclonal antibody mAb9 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.

[0048] The VL is 111 amino acids long. The number of amino acids in the four domains of its FR are 26, 17, 36 and 11, respectively. The number of amino acids in the three domains of LCDR are 6, 3 and 12, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-100aa, respectively. Their amino acid sequences are EDIESW (SEQ ID NO.1), DAS (SEQ ID NO.2) and QSAYASNSAGNT (SEQ ID NO.3), respectively.

[0049] The VH is 125 amino acids in length. The number of amino acids in the four domains of its FR are 25, 17, 38 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 7 and 19, respectively. HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 96aa-114aa, respectively. Their amino acid sequences are GFSLSSYA (SEQ ID NO.4), ISSSGST (SEQ ID NO.5) and ARGSFYPGYKTDFWDYFNL (SEQ ID NO.6), respectively.

[0050] 1. Detection Principle and Method A magnetic microparticle chemiluminescence immunoassay technique based on the double-antibody sandwich method was employed. Biotin-labeled E2 antibody was used to immobilize the biotinylated binding protein with SA magnetic beads. ALP was conjugated to the E2 complex using a sandwich method with rabbit monoclonal antibody. Simultaneously, E2, ALP substrate, and corresponding buffer components were placed on a Cosma fully automated magnetic microparticle chemiluminescence analyzer, and the instrument program was set for detection. A positive result was interpreted as a signal-to-noise ratio (SNR) greater than 2.0. The magnitude of the luminescence value reflects the amount of bound enzyme-labeled antibody and is directly proportional to the concentration of E2 in the sample. A standard curve was plotted based on the measured luminescence values ​​of the standards, as shown below. Figure 3 The E2 concentration value in the sample to be tested can be obtained from the standard curve.

[0051] 2. Composition of the magnetic particle chemiluminescence detection kit for E2 detection 1) SA magnetic beads bound to biotin-E2 binding protein: Take 50 μL of magnetic beads into a 0.5 mL centrifuge tube, place it on a magnetic rack, and remove the supernatant after 1 min; wash the magnetic beads 3 times with 0.5 mL of antibody dilution buffer; add a certain amount of biotin-labeled E2 binding protein and mix at room temperature for 60 min; after magnetic separation, resuspend in magnetic storage buffer at a working concentration of 0.5 mg / mL.

[0052] 2) mAb9 conjugation to ALP: First, the mAb9 antibody is reduced using 2-IT; then, an ALP-SMCC intermediate is formed; finally, ALP-SMCC is conjugated to the reducing antibody. After conjugation, the ALP is diluted to the working concentration using ALP preservation buffer.

[0053] 3) Washing buffer: Standard pH 7.4 PBST containing 0.05% Proclin 300, prepared as a 20-fold concentrate.

[0054] 4) Chemiluminescent colorimetric solution: purchased from Aivid Biotechnology.

[0055] 5) Sample diluent: PBST containing 1% BSA and 0.05% Proclin 300, filtered for sterilization.

[0056] 6) Standard: E2 small molecule, diluted to 5 μg / mL with PBS containing 1% BSA, 5% sucrose, 10% glycerol and 0.05% Proclin 300 as diluent, filtered for sterilization and aseptically dispensed.

[0057] 3. Testing of E2 clinical samples Clinical samples with different E2 concentrations were processed. Using E2 antibody as the coating antibody and mAb9 antibody as the detection antibody, the above detection method was applied to detect clinical samples of different concentrations. The results are shown below. Figure 4 .

[0058] Based on the results, the rabbit monoclonal antibody described in this application, used in a magnetic microparticle chemiluminescent immunoassay reagent, showed good correlation with clinical results within a sample range of 0-15000 pmol / L.

[0059] In summary, when the E2 sandwich rabbit monoclonal antibody mAb9 of this application is applied to immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence methods, the detection sensitivity of the double-antibody sandwich chemiluminescence platform for E2 standard antigen is less than 10 pg / mL. When the magnetochemiluminescence method is used to detect clinical samples, the clinical composite rate is >0.96 in the sample range of 0-15000 pmol / L, which is significantly higher than that of traditional competitive detection methods. Moreover, the process is simple and breaks through the limitations of traditional competitive methods.

[0060] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An E2 sandwich-type rabbit monoclonal antibody mAb9 or its antigen-binding fragment, characterized in that, Includes the light chain variable region VL and the heavy chain variable region VH. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.1 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

1. The amino acid sequence of LCDR2 is DAS or a variant with one amino acid substitution, deletion, or addition compared to DAS. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.3; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is the sequence shown in SEQ ID NO.4 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

4. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.5 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6 or a variant with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO.6; Furthermore, the E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment can specifically bind to E2 hormone.

2. The E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region VL comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.7, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids; the heavy chain variable region VH comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.8, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids.

3. The E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment according to claim 2, characterized in that, The light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.

8.

4. The E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

5. A biomaterial, characterized in that, The biomaterial is selected from polynucleotides, carriers, or cells. The polynucleotide encodes the E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment as described in any one of claims 1-4; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the vector, or is capable of expressing the E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment as described in any one of claims 1-4.

6. The method for preparing the E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment according to any one of claims 1-4, characterized in that, The method includes culturing the cells as described in claim 5, wherein the cells are prepared by transforming the cells with a polynucleotide encoding a rabbit monoclonal antibody mAb9 or an antigen-binding fragment thereof using the E2 sandwich method, wherein the polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the transformation includes co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

7. The application of the E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment according to any one of claims 1-4, or the biological material according to claim 5, characterized in that, The application is selected from one or more of the following: 1) Detection of E2 hormone for purposes other than disease diagnosis and treatment; 2) Prepare immunoassay products for detecting E2 hormone; 3) Used for purifying E2 hormone; 4) Prepare products for purifying E2 hormone.

8. The E2 hormone immunoassay product, characterized in that, The immunoassay product comprises the E2 sandwich rabbit monoclonal antibody mAb9 or its antigen-binding fragment as described in any one of claims 1-4, or the biological material as described in claim 5.

9. The E2 hormone immunoassay product according to claim 8, characterized in that, The E2 hormone immunoassay products are reagents or kits for detection using enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), immunoprecipitation, and magnetic microparticle chemiluminescence immunoassay.

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